N-glycosylation is a potent regulator of prion protein neurotoxicity.

The C-terminal domain of the cellular prion protein (PrPC) contains two N-linked glycosylation sites, the occupancy of which impacts disease pathology. In this study, we demonstrate that glycans at these sites are required to maintain an intramolecular interaction with the N-terminal domain, mediated through a previously identified copper-histidine tether, which suppresses the neurotoxic activity of PrPC. NMR and electron paramagnetic resonance spectroscopy demonstrate that the glycans refine the structure of the protein's interdomain interaction. Using whole-cell patch-clamp electrophysiology, we further show that cultured cells expressing PrP molecules with mutated glycosylation sites display large, spontaneous inward currents, a correlate of PrP-induced neurotoxicity. Our findings establish a structural basis for the role of N-linked glycans in maintaining a nontoxic, physiological fold of PrPC.

Obesity: a perfect storm for carcinogenesis.

Obesity-related cancers account for 40% of the cancer cases observed in the USA and obesity is overtaking smoking as the most widespread modifiable risk factor for carcinogenesis. Here, we use the hallmarks of cancer framework to delineate how obesity might influence the carcinogenic hallmarks in somatic cells. We discuss the effects of obesity on (a) sustaining proliferative signaling; (b) evading growth suppressors; (c) resisting cell death; (d) enabling replicative immortality; (e) inducing angiogenesis; (f) activating invasion and metastasis; (g) reprogramming energy metabolism; and (h) avoiding immune destruction, together with its effects on genome instability and tumour-promoting inflammation. We present the current understanding and controversies in this evolving field, and highlight some areas in need of further cross-disciplinary focus. For instance, the relative importance of the many potentially causative obesity-related factors is unclear for each type of malignancy. Even within a single tumour type, it is currently unknown whether one obesity-related factor consistently plays a predominant role, or if this varies between patients or, even in a single patient with time. Clarifying how the hallmarks are affected by obesity may lead to novel prevention and treatment strategies for the increasingly obese population.

Mechanisms of prion-induced toxicity.

Prion diseases are devastating neurodegenerative diseases caused by the structural conversion of the normally benign prion protein (PrPC) to an infectious, disease-associated, conformer, PrPSc. After decades of intense research, much is known about the self-templated prion conversion process, a phenomenon which is now understood to be operative in other more common neurodegenerative diseases such as Alzheimer's disease, Parkinson's disease, and amyotrophic lateral sclerosis. In this review, we provide the current state of knowledge concerning a relatively poorly understood aspect of prion diseases: mechanisms of neurotoxicity. We provide an overview of proposed functions of PrPC and its interactions with other extracellular proteins in the central nervous system, in vivo and in vitro models used to delineate signaling events downstream of prion propagation, the application of omics technologies, and the emerging appreciation of the role played by non-neuronal cell types in pathogenesis.

Bariatric surgery reveals a gut-restricted TGR5 agonist with anti-diabetic effects.

Bariatric surgery, the most effective treatment for obesity and type 2 diabetes, is associated with increased levels of the incretin hormone glucagon-like peptide-1 (GLP-1) and changes in levels of circulating bile acids. The levels of individual bile acids in the gastrointestinal (GI) tract after surgery have, however, remained largely unstudied. Using ultra-high performance liquid chromatography-mass spectrometry-based quantification, we observed an increase in an endogenous bile acid, cholic acid-7-sulfate (CA7S), in the GI tract of both mice and humans after sleeve gastrectomy. We show that CA7S is a Takeda G-protein receptor 5 (TGR5) agonist that increases Tgr5 expression and induces GLP-1 secretion. Furthermore, CA7S administration increases glucose tolerance in insulin-resistant mice in a TGR5-dependent manner. CA7S remains gut restricted, minimizing off-target effects previously observed for TGR5 agonists absorbed into the circulation. By studying changes in individual metabolites after surgery, the present study has revealed a naturally occurring TGR5 agonist that exerts systemic glucoregulatory effects while remaining confined to the gut.

Intrinsic toxicity of the cellular prion protein is regulated by its conserved central region.

The conserved central region (CR) of PrPC has been hypothesized to serve as a passive linker connecting the protein's toxic N-terminal and globular C-terminal domains. Yet, deletion of the CR causes neonatal fatality in mice, implying the CR possesses a protective function. The CR encompasses the regulatory α-cleavage locus, and additionally facilitates a regulatory metal ion-promoted interaction between the PrPC N- and C-terminal domains. To elucidate the role of the CR and determine why CR deletion generates toxicity, we designed PrPC constructs wherein either the cis-interaction or α-cleavage are selectively prevented. These constructs were interrogated using nuclear magnetic resonance, electrophysiology, and cell viability assays. Our results demonstrate the CR is not a passive linker and the native sequence is crucial for its protective role over the toxic N-terminus, irrespective of α-cleavage or the cis-interaction. Additionally, we find that the CR facilitates homodimerization of PrPC , attenuating the toxicity of the N-terminus.

Sleeve gastrectomy enhances glucose utilization and remodels adipose tissue independent of weight loss.

Sleeve gastrectomy (SG) induces weight loss-independent improvements in glucose homeostasis by unknown mechanisms. We sought to identify the metabolic adaptations responsible for these improvements. Nonobese C57BL/6J mice on standard chow underwent SG or sham surgery. Functional testing and indirect calorimetry were used to capture metabolic phenotypes. Tissue-specific glucose uptake was assessed by 18-fluorodeoxyglucose (18-FDG) PET/computed tomography, and RNA sequencing was used for gene-expression analysis. In this model, SG induced durable improvements in glucose tolerance in the absence of changes in weight, body composition, or food intake. Indirect calorimetry revealed that SG increased the average respiratory exchange ratio toward 1.0, indicating a weight-independent, systemic shift to carbohydrate utilization. Following SG, orally administered 18-FDG preferentially localized to white adipose depots, showing tissue-specific increases in glucose utilization induced by surgery. Transcriptional analysis with RNA sequencing demonstrated that increased glucose uptake in the visceral adipose tissue was associated with upregulation in transcriptional pathways involved in energy metabolism, adipocyte maturation, and adaptive and innate immune cell chemotaxis and differentiation. SG induces a rapid, weight loss-independent shift toward glucose utilization and transcriptional remodeling of metabolic and immune pathways in visceral adipose tissue. Continued study of this early post-SG physiology may lead to a better understanding of the anti-diabetic mechanisms of bariatric surgery.

Prions activate a p38 MAPK synaptotoxic signaling pathway.

Synaptic degeneration is one of the earliest pathological correlates of prion disease, and it is a major determinant of the progression of clinical symptoms. However, the cellular and molecular mechanisms underlying prion synaptotoxicity are poorly understood. Previously, we described an experimental system in which treatment of cultured hippocampal neurons with purified PrPSc, the infectious form of the prion protein, induces rapid retraction of dendritic spines, an effect that is entirely dependent on expression of endogenous PrPC by the target neurons. Here, we use this system to dissect pharmacologically the underlying cellular and molecular mechanisms. We show that PrPSc initiates a stepwise synaptotoxic signaling cascade that includes activation of NMDA receptors, calcium influx, stimulation of p38 MAPK and several downstream kinases, and collapse of the actin cytoskeleton within dendritic spines. Synaptic degeneration is restricted to excitatory synapses, spares presynaptic structures, and results in decrements in functional synaptic transmission. Pharmacological inhibition of any one of the steps in the signaling cascade, as well as expression of a dominant-negative form of p38 MAPK, block PrPSc-induced spine degeneration. Moreover, p38 MAPK inhibitors actually reverse the degenerative process after it has already begun. We also show that, while PrPC mediates the synaptotoxic effects of both PrPSc and the Alzheimer's Aß peptide in this system, the two species activate distinct signaling pathways. Taken together, our results provide powerful insights into the biology of prion neurotoxicity, they identify new, druggable therapeutic targets, and they allow comparison of prion synaptotoxic pathways with those involved in other neurodegenerative diseases.

PrP is a central player in toxicity mediated by soluble aggregates of neurodegeneration-causing proteins.

Neurodegenerative diseases are an enormous public health problem, affecting tens of millions of people worldwide. Nearly all of these diseases are characterized by oligomerization and fibrillization of neuronal proteins, and there is great interest in therapeutic targeting of these aggregates. Here, we show that soluble aggregates of α-synuclein and tau bind to plate-immobilized PrP in vitro and on mouse cortical neurons, and that this binding requires at least one of the same N-terminal sites at which soluble Aß aggregates bind. Moreover, soluble aggregates of tau, α-synuclein and Aß cause both functional (impairment of LTP) and structural (neuritic dystrophy) compromise and these deficits are absent when PrP is ablated, knocked-down, or when neurons are pre-treated with anti-PrP blocking antibodies. Using an all-human experimental paradigm involving: (1) isogenic iPSC-derived neurons expressing or lacking PRNP, and (2) aqueous extracts from brains of individuals who died with Alzheimer's disease, dementia with Lewy bodies, and Pick's disease, we demonstrate that Aß, α-synuclein and tau are toxic to neurons in a manner that requires PrPC. These results indicate that PrP is likely to play an important role in a variety of late-life neurodegenerative diseases and that therapeutic targeting of PrP, rather than individual disease proteins, may have more benefit for conditions which involve the aggregation of more than one protein.

Cellular prion protein targets amyloid-ß fibril ends via its C-terminal domain to prevent elongation.

Oligomeric forms of the amyloid-ß (Aß) peptide are thought to represent the primary synaptotoxic species underlying the neurodegenerative changes seen in Alzheimer's disease. It has been proposed that the cellular prion protein (PrPC) functions as a cell-surface receptor, which binds to Aß oligomers and transduces their toxic effects. However, the molecular details of the PrPC-Aß interaction remain uncertain. Here, we investigated the effect of PrPC on polymerization of Aß under rigorously controlled conditions in which Aß converts from a monomeric to a fibrillar state via a series of kinetically defined steps. We demonstrated that PrPC specifically inhibited elongation of Aß fibrils, most likely by binding to the ends of growing fibrils. Surprisingly, this inhibitory effect required the globular C-terminal domain of PrPC, which has not been previously implicated in interactions with Aß. Our results suggest that PrPC recognizes structural features common to both Aß oligomers and fibril ends and that this interaction could contribute to the neurotoxic effect of Aß aggregates. Additionally, our results identify the C terminus of PrPC as a new and potentially more druggable molecular target for treating Alzheimer's disease.

A Neuronal Culture System to Detect Prion Synaptotoxicity.

Synaptic pathology is an early feature of prion as well as other neurodegenerative diseases. Although the self-templating process by which prions propagate is well established, the mechanisms by which prions cause synaptotoxicity are poorly understood, due largely to the absence of experimentally tractable cell culture models. Here, we report that exposure of cultured hippocampal neurons to PrPSc, the infectious isoform of the prion protein, results in rapid retraction of dendritic spines. This effect is entirely dependent on expression of the cellular prion protein, PrPC, by target neurons, and on the presence of a nine-amino acid, polybasic region at the N-terminus of the PrPC molecule. Both protease-resistant and protease-sensitive forms of PrPSc cause dendritic loss. This system provides new insights into the mechanisms responsible for prion neurotoxicity, and it provides a platform for characterizing different pathogenic forms of PrPSc and testing potential therapeutic agents.

Identification of Anti-prion Compounds using a Novel Cellular Assay.

Prion diseases are devastating neurodegenerative disorders with no known cure. One strategy for developing therapies for these diseases is to identify compounds that block conversion of the cellular form of the prion protein (PrPC) into the infectious isoform (PrPSc). Most previous efforts to discover such molecules by high-throughput screening methods have utilized, as a read-out, a single kind of cellular assay system: neuroblastoma cells that are persistently infected with scrapie prions. Here, we describe the use of an alternative cellular assay based on suppressing the spontaneous cytotoxicity of a mutant form of PrP (Δ105-125). Using this assay, we screened 75,000 compounds, and identified a group of phenethyl piperidines (exemplified by LD7), which reduces the accumulation of PrPSc in infected neuroblastoma cells by >90% at low micromolar doses, and inhibits PrPSc-induced synaptotoxicity in hippocampal neurons. By analyzing the structure-activity relationships of 35 chemical derivatives, we defined the pharmacophore of LD7, and identified a more potent derivative. Active compounds do not alter total or cell-surface levels of PrPC, and do not bind to recombinant PrP in surface plasmon resonance experiments, although at high concentrations they inhibit PrPSc-seeded conversion of recombinant PrP to a misfolded state in an in vitro reaction (RT-QuIC). This class of small molecules may provide valuable therapeutic leads, as well as chemical biological tools to identify cellular pathways underlying PrPSc metabolism and PrPC function.

Synthetic Prions Provide Clues for Understanding Prion Diseases.

This Commentary highlights the article by Makarava et al that discusses the formation of synthetic prions and the role of substrate levels in their evolution.

Mechanisms of prion-induced neurodegeneration.

Transmissible spongiform encephalopathies (TSEs), or prion diseases, are fatal neurodegenerative disorders characterised by long incubation period, short clinical duration, and transmissibility to susceptible species. Neuronal loss, spongiform changes, gliosis and the accumulation in the brain of the misfolded version of a membrane-bound cellular prion protein (PrP(C)), termed PrP(TSE), are diagnostic markers of these diseases. Compelling evidence links protein misfolding and its accumulation with neurodegenerative changes. Accordingly, several mechanisms of prion-mediated neurotoxicity have been proposed. In this paper, we provide an overview of the recent knowledge on the mechanisms of neuropathogenesis, the neurotoxic PrP species and the possible therapeutic approaches to treat these devastating disorders.

A C-terminal membrane anchor affects the interactions of prion proteins with lipid membranes.

Membrane attachment via a C-terminal glycosylphosphatidylinositol anchor is critical for conversion of PrP(C) into pathogenic PrP(Sc). Therefore the effects of the anchor on PrP structure and function need to be deciphered. Three PrP variants, including full-length PrP (residues 23-231, FL_PrP), N-terminally truncated PrP (residues 90-231, T_PrP), and PrP missing its central hydrophobic region (Δ105-125, ΔCR_PrP), were equipped with a C-terminal membrane anchor via a semisynthesis strategy. Analyses of the interactions of lipidated PrPs with phospholipid membranes demonstrated that C-terminal membrane attachment induces a different binding mode of PrP to membranes, distinct from that of non-lipidated PrPs, and influences the biochemical and conformational properties of PrPs. Additionally, fluorescence-based assays indicated pore formation by lipidated ΔCR_PrP, a variant that is known to be highly neurotoxic in transgenic mice. This finding was supported by using patch clamp electrophysiological measurements of cultured cells. These results provide new evidence for the role of the membrane anchor in PrP-lipid interactions, highlighting the importance of the N-terminal and the central hydrophobic domain in these interactions.

The neuroprotective effect of Klotho is mediated via regulation of members of the redox system.

Generation of reactive oxygen species (ROS), leading to oxidative damage and neuronal cell death, plays an important role in the pathogenesis of neurodegenerative disorders, including Alzheimer disease. The present study aimed to examine the mechanism by which the anti-aging protein Klotho exerts neuroprotective effects against neuronal damage associated with neurodegeneration and oxidative stress. Pretreatment of rat primary hippocampal neurons and mouse hippocampal neuronal cell line HT22 with recombinant Klotho protected these cells from glutamate and oligomeric amyloid ß (oAß)-induced cytotoxicity. In addition, primary hippocampal neurons obtained from Klotho-overexpressing mouse embryos were more resistant to both cytotoxic insults, glutamate and oAß, compared with neurons from wild-type littermates. An antioxidative stress array analysis of neurons treated with Klotho revealed that Klotho significantly enhances the expression of the thioredoxin/peroxiredoxin (Trx/Prx) system with the greatest effect on the induction of Prx-2, an antioxidant enzyme, whose increase was confirmed at the mRNA and protein levels. Klotho-induced phosphorylation of the PI3K/Akt pathway, a pathway important in apoptosis and longevity, was associated with sustained inhibitory phosphorylation of the transcription factor forkhead box O3a (FoxO3a) and was essential for the induction of Prx-2. Down-regulation of Prx-2 expression using a lentivirus harboring shRNA almost completely abolished the ability of Klotho to rescue neurons from glutamate-induced death and significantly, but not completely, inhibited cell death mediated by oAß, suggesting that Prx-2 is a key modulator of neuroprotection. Thus, our results demonstrate, for the first time, the neuroprotective role of Klotho and reveal a novel mechanism underlying this effect.

A novel student-led approach to multiple-choice question generation and online database creation, with targeted clinician input.

PROBLEM: Multiple-choice questions (MCQs) are the main method of assessing medical student knowledge. As a result there is a high demand from medical students for formative MCQs. However, teaching staff rarely have the time or incentive to develop high-quality formative questions, focusing instead on material for high-stakes assessments. INTERVENTION: We have developed a novel student-led approach involving an interactive online question database, created by medical students for medical students. We adopted a staged approach to create an online bank of formative MCQ questions. First, students write MCQs following a standardized format. Questions are then peer-reviewed by other students, discussing relevant clinical topics, guidelines, and journals to improve question quality. The questions are then scrutinized by specialist doctors and academics. Next, questions are piloted online. Finally, question performance is evaluated statistically. This 5-stage student-led process produced a bank of more than 200 MCQs in three months. CONTEXT: This intervention was carried out by two final-year medical student leads at Cardiff University School of Medicine, UK. Final-year students were recruited to write and peer-review questions, and senior content specialists were recruited from the department. After piloting and evaluation of the questions, the question bank was made available as a learning resource to all medical students at Cardiff University. OUTCOME: Objective analysis of the created MCQs (discrimination indices and distractor analysis) indicated that the random sample of questions piloted were of high quality. When the questions were made available as online tests to approximately 600 students, usage data revealed that 2,800 tests were taken over a 3-month period, indicating that the resource was popular. In addition, subjective feedback from students question writers/reviewers was gathered via free text feedback forms and was invariably positive. We plan to continue the question generation process in Cardiff and would encourage other medical schools to adopt this approach. LESSONS LEARNED: Our 5-stage approach can generate a large volume of high-quality MCQs, addressing the demand from students for formative MCQ questions, with minimal teaching staff input. The project's benefits go beyond the creation of the resource, as involving students in the writing, review, and presentation of questions itself is useful pedagogically.

A mutant prion protein sensitizes neurons to glutamate-induced excitotoxicity.

Growing evidence suggests that a physiological activity of the cellular prion protein (PrP(C)) plays a crucial role in several neurodegenerative disorders, including prion and Alzheimer's diseases. However, how the functional activity of PrP(C) is subverted to deliver neurotoxic signals remains uncertain. Transgenic (Tg) mice expressing PrP with a deletion of residues 105-125 in the central region (referred to as ΔCR PrP) provide important insights into this problem. Tg(ΔCR) mice exhibit neonatal lethality and massive degeneration of cerebellar granule neurons, a phenotype that is dose dependently suppressed by the presence of wild-type PrP. When expressed in cultured cells, ΔCR PrP induces large, ionic currents that can be detected by patch-clamping techniques. Here, we tested the hypothesis that abnormal ion channel activity underlies the neuronal death seen in Tg(ΔCR) mice. We find that ΔCR PrP induces abnormal ionic currents in neurons in culture and in cerebellar slices and that this activity sensitizes the neurons to glutamate-induced, calcium-mediated death. In combination with ultrastructural and biochemical analyses, these results demonstrate a role for glutamate-induced excitotoxicity in PrP-mediated neurodegeneration. A similar mechanism may operate in other neurodegenerative disorders attributable to toxic, ß-rich oligomers that bind to PrP(C).

An N-terminal fragment of the prion protein binds to amyloid-ß oligomers and inhibits their neurotoxicity in vivo.

A hallmark of Alzheimer disease (AD) is the accumulation of the amyloid-ß (Aß) peptide in the brain. Considerable evidence suggests that soluble Aß oligomers are responsible for the synaptic dysfunction and cognitive deficit observed in AD. However, the mechanism by which these oligomers exert their neurotoxic effect remains unknown. Recently, it was reported that Aß oligomers bind to the cellular prion protein with high affinity. Here, we show that N1, the main physiological cleavage fragment of the cellular prion protein, is necessary and sufficient for binding early oligomeric intermediates during Aß polymerization into amyloid fibrils. The ability of N1 to bind Aß oligomers is influenced by positively charged residues in two sites (positions 23-31 and 95-105) and is dependent on the length of the sequence between them. Importantly, we also show that N1 strongly suppresses Aß oligomer toxicity in cultured murine hippocampal neurons, in a Caenorhabditis elegans-based assay, and in vivo in a mouse model of Aß-induced memory dysfunction. These data suggest that N1, or small peptides derived from it, could be potent inhibitors of Aß oligomer toxicity and represent an entirely new class of therapeutic agents for AD.

Sigma Receptor Ligands Are Potent Antiprion Compounds that Act Independently of Sigma Receptor Binding.

Prion diseases are invariably fatal neurodegenerative diseases of humans and other animals for which there are no effective treatment options. Previous work from our laboratory identified phenethylpiperidines as a novel class of anti-prion compounds. While working to identify the molecular target(s) of these molecules, we unexpectedly discovered ten novel antiprion compounds based on their known ability to bind to the sigma receptors, σ1R and σ2R, which are currently being tested as therapeutic or diagnostic targets for cancer and neuropsychiatric disorders. Surprisingly, however, knockout of the respective genes encoding σ1R and σ2R (Sigmar1 and Tmem97) in prion-infected N2a cells did not alter the antiprion activity of these compounds, demonstrating that these receptors are not the direct targets responsible for the antiprion effects of their ligands. Further investigation of the most potent molecules established that they are efficacious against multiple prion strains and protect against downstream prion-mediated synaptotoxicity. While the precise details of the mechanism of action of these molecules remain to be determined, the present work forms the basis for further investigation of these compounds in preclinical studies. Given the therapeutic utility of several of the tested compounds, including rimcazole and haloperidol for neuropsychiatric conditions, (+)-pentazocine for neuropathic pain, and the ongoing clinical trials of SA 4503 and ANAVEX2-73 for ischemic stroke and Alzheimer's disease, respectively, this work has immediate implications for the treatment of human prion disease.

New role of fat-free mass in cancer risk linked with genetic predisposition.

Cancer risk is associated with the widely debated measure body mass index (BMI). Fat mass and fat-free mass measurements from bioelectrical impedance may further clarify this association. The UK Biobank is a rare resource in which bioelectrical impedance and BMI data was collected on ~ 500,000 individuals. Using this dataset, a comprehensive analysis using regression, principal component and genome-wide genetic association, provided multiple levels of evidence that increasing whole body fat (WBFM) and fat-free mass (WBFFM) are both associated with increased post-menopausal breast cancer risk, and colorectal cancer risk in men. WBFM was inversely associated with prostate cancer. We also identified rs615029[T] and rs1485995[G] as associated in independent analyses with both PMBC (p = 1.56E-17 and 1.78E-11) and WBFFM (p = 2.88E-08 and 8.24E-12), highlighting splice variants of the intriguing long non-coding RNA CUPID1 (LINC01488) as a potential link between PMBC risk and fat-free mass.